New Approaches for Probing PARylation in living Cells

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Poly(ADP-ribos)ylation (PARylation) is a major post-translational modification and signalling event in most eukaryotes. Fundamental processes like DNA repair and transcription are coordinated by this transient polymer and it’s binding to proteins. ADP-ribosyltransferases (ARTs) build complex ADP-ribose chains from NAD+ onto various acceptor proteins. The complexity of the modification and the absence of suitable tools render molecular studies of PARylation challenging. Building on our successful earlier work that already lead to several joint publications, the major aim of this collaborative project is to establish means for quantitative studies of DNA photodamage induced protein PARylation dynamics in live cells with high temporal and spatial resolution. Our approach is based on the development of novel synthetic NAD+-based tools and advanced optical microscopy techniques. Based on the preliminary results, the Marx group will develop means to deliver modified NAD+-analogues through the cell membrane that do not interfere with the cell viability. Two kinds of modified NAD-analogues are envisioned that will allow imaging (through modification with a fluorescent dye) and affinity enrichment of protein that are modified through processing of desthiobiotin-modified NAD+-analogues. The Zumbusch group will further develop optical experiments to optimize NIR microirradiation such that controlled induction of specific types of DNA damages becomes possible. To this end, we will establish means to independently control pulse energy and average power of the NIR laser used for microirradiation. At the same time, experimental protocols for the application of synthetic NAD+-based tools and optical microscopy of PAR formation with minimized effects on cell health will be investigated. These will be evaluated by live cell microscopy of PARylation dynamics of selected proteins after DNA photo-damage and path the way for future applications.